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. 2025 May 24;119:107398. doi: 10.1016/j.ultsonch.2025.107398

Visibly acoustic delivery of IR808 into tumor via gas vesicles enhances photothermal therapy efficacy against tumor☆

Xin Meng a,b, Yanan Feng a, Yuanyuan Wang e, Chen Lin a, Wei Wang a, Sutian Zhu a, Bing Guo c, Huaiyu Wang d, Litao Sun a,⁎, Fei Yan e,⁎
PMCID: PMC12166705  PMID: 40446792

Abstract

Photothermal therapy (PTT) has emerged as a promising new approach in tumor treatment, with the great advantages including non-invasiveness and temporal controllability. However, the effective delivery of photothermal agents into tumor remains a significant challenge, limiting its clinical translational application. In this study, we developed a kind of photothermal agents modified with gas vesicles (GVs), greatly facilitating ultrasound/fluorescence imaging-guided delivery of photothermal agents and enhancing the efficacy of photothermal therapy. The GVs were synthesized and extracted from Halobacterium NRC-1, followed with modification with IR808. The resulting GVs-IR808 were able to be visually tracked by ultrasound and fluorescence imaging. Upon their arrival at the tumor area after systemic administration, ultrasound irradiation was applied to induce the cavitation of GVs-IR808, greatly promoting IR808 delivery into the tumor. The subsequent laser irradiation was applied and resulted in a significant inhibition of tumor growth. In conclusion, our study provides a novel approach for ultrasound/fluorescence dual-modal imaging-guided photothermal treatment of breast tumors.

Keywords: Photothermal therapy, IR808, Gas vesicles, Drug delivery, Breast tumor

1. Introduction

Photothermal therapy (PTT) is a new type of treatment that uses photothermal agents to convert light energy into heat energy, killing tumor cells by raising the local temperature. In this strategy, photothermal agents (PTAs) need to be delivered into the tumor tissue through the bloodstream and near infrared (NIR) light is then applied for increasing the local temperature of the tumor tissue, producing the coagulative necrosis of tumor cells [1,2]. However, in most solid tumors, the use of photothermal therapy is challenged by the heterogeneity and the high interstitial pressure of the tumor tissue, resulting in limited concentration of drugs in the tumor cells. Due to these limitations, PTT-induced anti-tumor strategies often fail to completely eradicate tumors, leading to tumor recurrence and metastasis. Therefore, it is highly desirable to increase the delivery efficiency of PTAs into tumor in order to maximize their efficacy.

Recently, the ultrasound cavitation effect has gradually become one of the cutting-edge technologies to facilitate drug delivery. The mechanism of ultrasound-enhanced drug delivery is mainly based on its acoustic cavitation, an effect generated by the vibration of the acoustic agents excited by the ultrasound wave, which can enhance the drug delivery efficiency through the sonoporation effect from the mechanical forces on vascular endothelial cells, enlarging the gap between vascular endothelial cells and promoting the penetration and absorption of the drugs [3]. At the same time, the mechanical forces generated by the ultrasound cavitation effect can also perforate the cell membranes and create small temporary microscale holes on cells, which can facilitate the uptake of drugs into cells [[4], [5], [6]]. Especially, the drugs can also be bound onto the surface of bubbles or encapsulated into the bubbles, preventing the premature release of drugs at the undesired time and place [7,8]. Numerous literatures demonstrated the enhanced drug delivery into tumor through ultrasound cavitation effect. For example, Shakeri-Zadeh et al delivered nanocapsules containing 5-fluorouracil (5-Fu) into mouse colon tumor through ultrasound. After intravenous injection of the nanocapsules, ultrasound irradiation was applied and increased the accumulation of nanoparticles in the tumor tissue [9]. Oda et al used the ultrasound cavitation to promote cell transfection and introduce tumor-associated antigenic peptides into dendritic cells (DCs), efficiently delivering tumor antigens into DC cells and inhibiting melanoma metastasis [10].

Microbubbles are the important factor for the generation of ultrasound cavitation effect. When microbubbles are exposed to ultrasound with adequate energy, these microbubbles collapses and releases intense energy for a short time, resulting in the generation of cavitation effects such as microstreaming, microjet, shock wave, thermal effect and sonoluminescence, etc [11,12]. However, microbubbles have microscale particle size and are confined within the blood vessel, greatly decreasing the effects of cavitation-enhanced drug delivery. Unlike microbubbles, gas vesicles (GVs) from Halobacterium NRC-1 have about 200 nm particle size. These nanobubbles can pass through the tumor blood vessel and enhance the delivery of nucleic acid drugs into tumors via the acoustic cavitation effects [[13], [14], [15]]. In this study, we isolated GVs from Halobacterium NRC-1 and modified them with IR808 to obtain GVs-IR808 for ultrasound/fluorescence imaging-guided PTT tumor therapy. Considering that there is a gap of about 380–780 nm between endothelial cells in tumor blood vessels, the nanoscale GVs can penetrate of tumor vessels via enhanced permeability and retention (EPR) effect after systemic administration of GVs-IR808. Interestingly, these GVs-IR808 could be tracked by ultrasound/fluorescence imaging. Upon the utilization of ultrasound irradiation, cavitation effects would be induced and delivered IR808 into the tumor cells, greatly enhancing the anti-tumor efficacy and prolonging the survival time (Fig. 1).

Fig. 1.

Fig. 1

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Schematic illustration of the preparation of GVs-IR808, their acoustic delivery into tumor and photoacoustic/fluorescence imaging-guided photothermal therapy of breast cancer. GVs were extracted from Halobacterium NRC-1 (Halo) and modified with IR808 to obtain GVs-IR808. After systemic administration, GVs-IR808 can be visualized by ultrasound/fluorescence dual-modal imaging and the enhanced delivery of IR808 into the tumor can be achieved through the cavitation effect, greatly improving the anti-tumor efficacy of photothermal therapy.

2. Materials and Methods

2.1. Materials

IR808 (1-(5-Carboxypentyl)-2-[2-[3-[[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene]ethylidene]-2-chloro-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-3H-indolium bromide) was provided by Harbin Institute of Technology, Shenzhen. 1-ethyl-(3-dimethyl aminopropyl) carbamide (EDC), and N-hydroxy succinimide (NHS) were obtained from Shanghai Macklin Biochemical Co. Ltd. Dimethyl sulfoxide (DMSO) was obtained from Xi’an Ruixi biological technology Co.Ltd. The mouse breast cancer cells (4T1) were obtained from American Type Culture Collection. Dulbecco’s modified Eagle’s medium (DMEM) culture medium, fetal bovine serum (FBS), penicillin and streptomycin solution, trypsin containing 0.25 % EDTA were purchased from Gibco (USA). Calcein-AM/PI double strain kit, CCK-8 assay kit and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Beyotime Institute of Biotechnology.

2.2. Preparation of GVs-IR808

Halobacterium NRC-1 were cultivated in ATCC culture medium (250 g NaCl, 20 g MgSO4, 3 g Trisodium citrate, 2 g KCl, 3 g Yeast extract, 5 g Tryptone, 1 L purified water)at 37 °C with 220 rpm/min for 2 weeks. The bacteria were then kept in a separation funnel for 1–2 weeks to collect the floated bacteria. These bacteria were lysed by using TMC lysis buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 2 mM CaCl2, pH 7.5) and then separated by centrifuging at 300g for 4 h to obtain GVs. The GVs are further purified by washing for 3 to 4 times with phosphate-buffered saline (PBS) and centrifuging at 250g for 4 h. The concentration of GVs was determined at a wavelength of 500 nm by using a microplate reader (Multiscan GO, Thermo Scientific, Waltham, MA, United States).

To conjugate IR808 to the surface of the GVs, an amidation reaction was performed in the presence of EDC and NHS. Briefly, IR808 was dispersed in DMSO with EDC (7 mg) and NHS (10 mg) at given concentrations. This mixture was incubated at room temperature for 3 h and then diluted with pure water. This diluted solution was slowly added to GVs solution (in PBS) and further incubated overnight. The reaction solution was centrifuged, and then washed with PBS for 4 times to remove free EDC, NHS and IR808.

2.3. Characterization of GVs-IR808

The morphologies of GVs-IR808 were observed by transmission electron microscopy (TEM) (Hitachi H-7650, Japan). The particle size and zeta potential of GVs and GVs-IR808 were measured using a Zetasizer analyzer (Malvern, United Kingdom), with each sample measured in triplicate. The absorbance spectra of IR808 at 12.5, 25, 50, 100, 150 μg/mL concentrations were determined by UV–Vis spectrophotometer. The fluorescence spectra of GVs, IR808 and GVs-IR808 are measured by a fluorometer with an excitation wavelength of 808 nm.

To determine the amount of IR808 in GVs-IR808, the IR808 solution at 0–160 μg/mL was prepared with pure water and DMSO (3:1), and the absorbance of the IR808 at 808 nm was measured by UV–Vis spectroscopy to plot the standard curve of the IR808. Different concentrations of IR808 were coupled with GVs, and the GVs-IR808 solution was obtained after centrifugation to remove free EDC, NHS and IR808. A clear solution containing free IR808 was obtained after destructing these GVs in the GVs-IR808 solution by ultrasonic irradiation. The content of IR808 in GVs-IR808 was calculated using a standard curve of IR808. The conjugation efficiency of IR808 was calculated using the following formula: conjugation efficiency (%) = (Free IR808 content / initial IR808 content) × 100 %.

2.4. In vitro photothermal effect and photothermal stability

The in vitro photothermal effect of GVs-IR808 was observed by irradiating different concentrations of IR808 solution (0, 20, 40, 80 or 160 µg/mL) or GVs-IR808 solution with 808 nm laser at a power density of 1.0 W/cm2 for 5 min at room temperature. In addition, 160 µg/mL GVs-IR808 solution were irradiated with different power densities of the 808 nm laser. The distance between sample and laser was fixed at 10 cm. An infrared thermal camera (FLIR E4) was applied to monitor the temperature at different irradiation time. The temperature was recorded at 30 s interval. Moreover, 160 µg/mL GVs-IR808 were irradiated at 1.0 W/cm2 laser for 5 min of IR808 in GVs-IR808 to assess the photothermal stabilities of GVs-IR808. The laser was turned off and cooled naturally to room temperature, and then repeated 5 Laser ON/OFF cycles for temperature monitoring.

2.5. Calculation of photothermal conversion efficiency

The photothermal conversion efficiency (η) of GVs-IR808 was determined by Roper’s method and calculated by the following equations:

η = (hS (Tmax – Tsur)–QDis)/(I(1 – 10-A808nm) (1)

where h is the thermal conductivity coefficient; S is the surface area of the container, I is the laser power at 1.0 W/cm and A808 nm is the absorbance of GVs-IR808. Tmax is the maximum equilibrium temperature (51.5 °C); Tsur is the surrounding ambient temperature (21.1 °C). QD is the heat dissipated from the light absorbed by the container and approximately equal to zero in this system. The value of hS is derived from Equation:

τs = mc/hS (2)

Where m is the mass of water, and c is the heat capacity of deionized water (4.2 Cp (J g-1)), and the value of τs is calculated according to Equations (3), (4) as follows:

t = − τs ln(θ) (3)
θ = T − Tsur/Tmax − Tsur (4)

The time constant for thermal conductivity was calculated to be τs = 118.2 s for GVs-IR808 by applying the linear time data from the cooling stage versus the negative natural logarithm of the driving force temperature.

2.6. Cytotoxicity of GVs-IR808

The murine breast cancer 4T1 cells line and Hela cells were cultured in the Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 1 % penicillin/streptomycin and 10 % FBS at 37 °C under 5 % CO2 atmosphere. To evaluate the cytotoxicity of GVs-IR808, 4T1 and Hela cells were seeded into a 96-well plate at a density of 1 × 104 per well overnight. After cell adhesion, cell medium was replaced with a new medium containing given amount of GVs-IR808. After 12 h co-incubation, cell viabilities were tested by the standard CCK-8 assay based on the absorbance at the wavelength of 450 nm via a multi-mode microplate reader.

2.7. Cell uptake assay

4T1 cells were seeded in confocal dishes and cultured to a density of about 70 % and randomly divided into three groups: the control group without any treatment (Control), non-ultrasonic cavitation group (US -) and ultrasonic cavitation group (US +). The ultrasonic cavitation was performed with 1.0 MHz transducer, at 1.0 W/cm2, 20 % duty cycle for 60 s, and the culture medium was removed and rinsed with PBS. Cells were fixed with 4 % paraformaldehyde for 15 min, stained with DAPI for 5 min and then washed 3 times in PBS, followed by observation using CLSM. These treated cells were also analyzed by flow cytometry.

2.8. In vitro anti-tumor effects

4T1 cells seeded in 96-well culture plates at a density of 1 × 104 cells per well were divided into six groups: the control group without any treatment (Control), US combined with Laser irradiation group (US + Laser), GVs-IR808 group (GVs-IR808), GVs-IR808 combined with US group (GVs-IR808 + US), GVs-IR808 combined with Laser irradiation group (GVs-IR808 + Laser) and GVs-IR808 combined with US and Laser irradiation group (GVs-IR808 + US + Laser). For the control group, cells were treated with serum-free culture medium. For US + Laser group, cells were irradiated by US (1.0 MHz, 1.0 W/cm2, 20 % duty cycle) for 1 min and Laser (808 nm and 1.0 W/cm2) for 5 min in the absence of GVs-IR808. For GVs-IR808 group, cells were exposed to GVs-IR808 and immediately rinsed with PBS (IR808 concentration of 160 μg/mL).For GVs-IR808 + US group, cells were treated with GVs-IR808 and then irradiated by US (1.0 MHz, 1.0 W/cm2, 20 % duty cycle) for 1 min, followed by rinse with PBS. For GVs-IR808 + Laser group, cells were exposed to GVs-IR808 and immediately rinsed with PBS, and then irradiated by Laser (808 nm and 1.0 W/cm2) for 5 min. For GVs-IR808 + US + Laser group, cells were exposed to GVs-IR808 and were irradiated by US (1.0 MHz, 1.0 W/cm2, 20 % duty cycle) for 1 min, followed by rinse with PBS, and then irradiated by Laser (808 nm and 1.0 W/cm2) for 5 min. Cell viabilities were tested by the standard CCK-8 assay. Meanwhile, cells in different groups were also stained with Calcein-AM and PI solutions and observed by the inverted fluorescent microscope, respectively.

2.9. In vitro ultrasound imaging

The in vitro imaging phantoms were prepared with 1 % agarose in pure water. GVs-IR808 was added to 1 % agarose wells (200 μL per sample). Imaging was performed using the L11-3U linear array transducer ultrasound diagnostic device (Mindray Resona R9T, Mindray, Shenzhen, China) in B mode and contrast mode, the ultrasonic probe was placed directly one side of the agarose and then burst the bubbles. The parameters were kept as follows: acoustic power: 5.13 %, mechanical index: 0.013, contrast gain: 65 db. After manually defining the region of interest (ROI), the pre- and post-burst signals were quantitatively analyzed using image J software.

2.10. Breast tumor model

Female Balb/c mice with the age of 4–6 weeks were purchased from Baitantong Biotechnology Co. (Zhuhai, China). Mouse studies followed the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and the protocols were approved by the Committee on the Ethics of Animal Experiments at the Shenzhen Institutes of Advanced Technology (SIAT) of the Chinese Academy of Science (CAS). 4T1 tumor-bearing mouse model was established by subcutaneously injecting 4T1 cells (1 × 106) into the right flank of each female BALB/c mice (about 20 g). Treatment was initiated when the mean tumor volume reaches about 80–100 mm3. The tumor volume is calculated as follows: length × width2/2.

2.11. In vivo ultrasound contrast imaging

GVs-IR808 (100 μL at OD3.0) were injected into mice (n = 3) via the tail vein. Ultrasound imaging were performed using a 3.0–11.0 MHz line array transducer equipped in the Mindray Resona R9T. All parameters were kept as follows: sound power: 5.13 %, mechanical index: 0.145, contrast gain: 70 dB in the imaging procedure. Burst pulses were applied to collapse the GVs-IR808 when the contrast signal achieved the peak value and repeated five times. This step was repeated five times at ten second intervals and repeated. The built-in software in the device was used for quantitatively analyzeing the contrast acoustic signals.

2.12. In vivo fluorescence imaging and infrared thermography

To evaluate the efficiency of acoustic delivery of IR808 into the tumor, tumor-bearing mice were randomly divided into two groups: the GVs-IR808 group, and the GVs-IR808 combined with acoustic irradiation (GVs-IR808 + US). The dose of IR808 administered to each mouse was kept at 160 µg. The fluorescence images of the tumor region were recorded at different times (0 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h) using a fluorescence system (Spectrum, Caliper, USA). In addition, the relative fluorescence intensities of the tumor region and major organs were measured. Sections of the tumors from 24 treated mice were prepared and stained with DAPI for detection of IR808 by fluorescence microscopy. The temperature changes were also detected in the tumors received with acoustic delivery of IR808. Briefly, GVs-IR808 (IR808 concentration at 160 µg/each) or equal volume of PBS were systemically administered in 4T1 tumor-bearing mice. After the tumors were irradiated by ultrasound for inducing the cavitation of GVs-IR808 and acoustic delivery of IR808, 808 nm laser irradiation at 1.0 W/cm2 then applied to the tumors for 10 min. The temperature changes and NIR thermal images of tumor region were recorded by an infrared thermal imaging camera (FLIR E4).

2.13. In vivo anti-tumor efficacy of GVs-IR808

4T1 tumor-bearing mice were randomly divided into six groups (n = 5) when the tumor volume reached approximately 80–100 mm3, including: the control group without any treatment (Control), US combined with Laser irradiation group (US + Laser), GVs-IR808 group (GVs-IR808), GVs-IR808 combined with US group (GVs-IR808 + US), GVs-IR808 combined with Laser irradiation group (GVs-IR808 + Laser) and GVs-IR808 combined with US and Laser irradiation group (GVs-IR808 + US + Laser). Mice in the control and US + Laser groups were intravenously injected with PBS. Mice in the GVs-IR808, GVs-IR808 + US, GVs-IR808 + Laser, and GVs-IR808 + US + Laser groups were injected of GVs-IR808 (IR808 concentration at 160 µg for each mouse). Mice in the GVs-IR808 + US group were received with acoustic irradiation for 5 min. Mice in the GVs-IR808 + Laser group were received with laser irradiation at 808 nm (1.0 W/cm2) at 24 h after bubble injection for 10 min. Mice in the US + Laser groups and the GVs-IR808 + US + Laser group were received with acoustic irradiation for 5 min after bubble injection and irradiated with an 808 nm laser at 1.0 W/cm2 for 10 min after 24 h. The body weights and tumor volume of the mice were recorded every other day after treatment. Mice were sacrificed when the tumor volume was more than 2000 mm3.

2.14. Blood and histological analysis

Healthy Balb/c mice were randomly divided into six groups (n = 5): the control group without any treatment (Control), US combined with Laser irradiation group (US + Laser), GVs-IR808 group (GVs-IR808), GVs-IR808 combined with US group (GVs-IR808 + US), GVs-IR808 combined with Laser irradiation group (GVs-IR808 + Laser) and GVs-IR808 combined with US and Laser irradiation group (GVs-IR808 + US + Laser). After 14 days of treatment, all mice were sacrificed. Blood samples were collected for biochemical analyses, and major organs such as heart, liver, spleen, lungs and kidneys were removed for HE staining. Three healthy mice were employed as the control group without any treatment. The blood samples were collected for biochemistry analysis and major organs including heart, liver, spleen, lung, and kidney were harvested for H&E staining.

2.15. Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8 software. The data were presented as the mean value ± standard deviation (SD).

3. Result

3.1. Synthesis and Characterization of GVs-IR808

GVs-IR808 were synthesized through covalently conjugating IR808 onto the surface of GVs extracted from Halobacterium NRC-1. As shown in transmission electron microscopy (TEM) images, the synthesized GVs-IR808, similar with GVs, had a relatively monodisperse structure with a spindle shape, and the hydrodynamic particle size of GVs-IR808 was slightly larger than that of the plain GVs, with 197 nm average particle size (Fig. 2A). The zeta potential of GVs-IR808 was −36.27 ± 1.05 mV, significantly lower than the plain GVs at −28.76 ± 0.93 mV (Fig. 2B). UV absorbance of GVs-IR808 were analyzed, showing that the characteristic peaks of GVs-IR808 appeared at 808 nm, consistent with IR808 (Fig. 2D–F). In addition, fluorescence spectrometer analysis showed that the maximum emission peak of GVs-IR808 was similar to that of IR808 (Fig. 2C). Furthermore, we found that the higher concentration of IR808 was used, the more IR808 would be conjugated onto the GVs. Through developing the linear relationship of light absorption range of IR808 at different concentrations, we determined the conjugation efficiency of IR808 in GVs-IR808 could achieve 43 % (Fig. S1). These results showed that IR808 was successfully conjugated to the surface of GVs.

Fig. 2.

Fig. 2

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Characterization of GVs-IR808. (A) Size distribution of GVs and GVs-IR808. The inset shows the TEM image of GVs. The scale bars are 500 nm; (B) Zeta potential of IR808, GVs and GVs-IR808;(C, D) Fluorescence and UV–vis spectra of GVs, IR808 and GVs-IR808;(E, F) UV–Vis spectra of IR808 and GVs-IR808 at different concentrations.

3.2. In vitro photothermal performance and stability of GVs-IR808

In order to evaluate the photothermal performance of GVs-IR808, we used 808 nm near-infrared laser to irradiate the plain GVs, IR808 or GVs-IR808 (with the equivalent of IR808) at 1.0 W/cm2 for 5 min. The results showed that the temperature elevation (ΔT) from GVs-IR808 sample achieved 30 °C, achieving a comparable ΔT with the free IR808 (31.7 °C). By contrast, no obvious temperature elevation was observed for the plain GVs under 808 nm laser irradiation (Fig. 3A, S2). Also, GVs-IR808 had IR808 concentration-dependent photothermal properties, achieving the most significant ΔT (30 °C) for GVs-IR808 with 160 μg/mL IR808 concentration, whereas only lightly increased ΔT (0.7 °C) was observed for the deionized water under the same irradiation condition (Fig. 3B, S3). Similarly, the temperature elevation of GVs-IR808 was recorded at different laser powers (0.75 W/cm2, 1.0 W/cm2, 1.25 W/cm2, 1.5 W/cm2) when keeping IR808 concentration at 160 μg/mL (Fig. 3C, S4), revealing that the ΔT of GVs-IR808 increased significantly along with the increase of NIR irradiation power. Furthermore, the photothermal stability of GVs-IR808 was also assessed through repeated exposure to NIR laser for five laser on/off cycles (808 nm, 1.0 W/cm2, 5 min each). Fig. 3D clearly showed that GVs-IR808 could maintain the same level temperature elevation, suggesting the excellent photostability for GVs-IR808. As showed in the Fig. 3E, the temperature equilibrium time during laser on/off process reached to 5 min. Generally, photothermal conversion efficiency (η) is considered as the gold standard for evaluating the photothermal capability for PTT. Therefore, the η of GVs-IR808 was calculated according to the fitting curve of time versus −Ln(θ) derived from the cooling stage (Fig. 3F). The η in this study was quantified to be 41 %.

Fig. 3.

Fig. 3

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The photothermal performance and stability of GVs-IR808. (A) Plot of temperature elevation (ΔT) of GVs-IR808 (IR808 concentration: 160 μg/mL), IR808 (160 μg/mL) and GVs suspension under NIR irradiation (808 nm, 1.0 W/cm2, 5 min). (B) Plot of temperature elevation (ΔT) of deionized water and GVs-IR808 suspension at different concentrations (20, 40, 80 and160 μg/mL) under NIR irradiation (808 nm, 1.0 W/cm2, 5 min). (C) Plot of temperature elevation (ΔT) of GVs-IR808 (IR808 concentration: 160 μg/mL) suspension under different power densities of NIR irradiation (808 nm, 0.75, 1.0, 1.25 and 1.5 W/cm2, 5 min). (D) Plot of temperature elevation (ΔT) of GVs-IR808 (IR808 concentration: 160 μg/mL) irradiated by an 808 nm laser (1.0 W/cm2) for five irradiation cycles. (E) Plot of temperature elevation (ΔT) of GVs-IR808 (IR808 concentration: 160 μg/mL) responded to the 808 nm laser (1.0 W/cm2) on/off. (F) Linear time data versus −Ln(θ) obtained from the cooling stage after NIR laser off.

3.3. In vitro photothermal anti-tumor effects of GVs-IR808

Before testing, the cytotoxic effects of GVs-IR808 at different concentrations were firstly examined by the CCK-8 assay on 4T1 and Hela cells. After 12 h incubation of GVs-IR808 with 4T1 and Hela cells, GVs-IR808 showed negligible toxicity to both types of cells at all the tested concentrations (IR808 concentration at 0–200 μg/mL) (Fig. 4A). To evaluate whether the ultrasonic cavitation effect could promote the delivery of IR808 into 4T1 cells, GVs-IR808 were added into the culture medium and followed by ultrasound irradiation. As showed in Fig. 4B and 4C, the GVs-IR808 + US group showed strong red fluorescent signals in the cells but hardly signals could be observed in the non-irradiated GVs-IR808 group. Similarly, the fluorescence intensities of cells in the GVs-IR808 group and the GVs-IR808 + US group were quantitatively assessed by flow cytometry (Fig. 4D, E), and the results showed significantly stronger fluorescence signal in the GVs-IR808 + US group after ultrasound irradiation, achieving 6.48-fold higher fluorescence signal intensity vs non-irradiated GVs-IR808 group. To investigate the in vitro photothermal anti-tumor effects of GVs-IR808, these US-irradiated or non-irradiated 4T1 cells were further received with laser irradiation. Calcein AM/PI staining revealed that almost all cells treated with GVs-IR808 + US + Laser were killed (red fluorescence, Fig. 4G). As seen in Fig. 4F, the cell viability of the untreated control and GVs-IR808 groups remained above 90 %, just like the US + Laser group and GVs-IR808 + US group. By contrast, the GVs-IR808 + Laser and GVs-IR808 + US + Laser groups exhibited stronger cytotoxic effects on 4T1 cells. Notably, the GVs-IR808 + US + Laser group showed the strongest cytotoxic effect on 4T1 cells, with the lowest cell survival. All of these data proved GVs-IR808 had good cytotoxicity against 4 T1 cells when exposed to US and Laser irradiation.

Fig. 4.

Fig. 4

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In vitro anti-tumor effects of GVs-IR808. (A) Cytotoxicity of GVs-IR808 to Hela and 4T1 cells without US and Laser irradiation. (B, C) Fluorescence images and quantitative analysis of ultrasound irradiated (US + ) and non-irradiated (US -) 4T1 cells stained with DAPI. Scale bar = 100 nm. (n = 3, US + vs. US-, ***p < 0.001; US + vs. Control, ***p < 0.001). (D, E) Flow cytometry analysis of fluorescence and quantification of ultrasound irradiated (US + ) and non-irradiated (US -) 4T1 cells. (n = 3, US + vs. US-, ****p < 0.0001; US + vs. Control, ****p < 0.0001) (F) The fluorescence images of the above treated cells stained with Calcein AM/PI assay. Scale bar = 100 µm. (G) The viability of cells treated with or without GVs-IR808 exposed to US (1.0 W/cm2, 5 min) and/or laser (1.0 W/cm2, 5 min). Here, U refers to ultrasound, and L refers to laser treatment.

3.4. The imaging and delivery performance of GVs-IR808

In this study, we firstly examined the imaging ability of GVs-IR808 in 1 % agar phantom by using ultrasound imaging machine equipped with a clinical line array transducer, and the contrast signal of GVs-IR808 was detected at a concentration of GVs at OD500 2.0. Clear contrast signals could be observed and these signals would disappear upon the burst was performed (Fig. 5A, B). To verify whether the ultrasound cavitation effect can improve the delivery of IR808, GVs-IR808 at OD500 3.0 were administrated into the tail vein of 4 T1 tumor-bearing mice, significant signal acoustic enhancement could be immediately observed at the tumor site. After that, ultrasound irradiation was applied to the tumor. Fig. 5C clearly showed that these contrast signals disappeared immediately and gradually reappeared at the tumor site due to the reperfusion of GVs-IR808. Interestingly, when the burst was repeated for five times, the disappearance and appearance of the contrast signals could be still observed (Fig. 5D). After ultrasound irradiation, we further examined the fluorescence imaging performance of GVs-IR808 in the tumor area. As shown in Fig. 5E, we can see that the fluorescence intensity in the tumor of mice received with GVs-IR808 gradually increased to a peak level at 24 h and then decayed away. By contrast, fast increased fluorescence signals could be observed in the tumor from 4 h post acoustic irradiation, achieving a peak at 12 h. The fluorescence signal intensity of GVs-IR808 + US in the tumor was significantly higher than the tumor of mice received with GVs-IR808 (Fig. 5F). Fig. 5G and 5H showed most of IR808 was accumulated in the liver for GVs-IR808 group but in the tumor for GVs-IR808 + US group. Histological analysis of tumor further confirmed significantly stronger red fluorescent signals could be observed in the GVs-IR808 + US group than that of GVs-IR808 group (Fig. 5I). These results suggest that ultrasound cavitation of GVs-IR808 can be visualized by ultrasound and significantly enhance the delivery of IR808 into the tumor.

Fig. 5.

Fig. 5

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The imaging and delivery performance of GVs-IR808. (A, B) The in vitro ultrasound imaging of GVs-IR808 at B-mode and contrast mode and their contrast signal intensity before and after burst (n = 3, ***p < 0.001); (C) B-mode and contrast images of the GVs-IR808 in the tumor region in 5 short-pulsed burst (two bursts were separated by about 10 s); (D) Time-intensity curves of GVs-IR808 contrast signals during 5 short-pulsed bursts; (E,F) In vivo fluorescence imaging of tumor-bearing mice and signal intensity analysis of tumors after intravenous injection of GVs-IR808 at different time intervals. (n = 3, p < 0.05); (G, H) Ex vivo fluorescence imaging and fluorescence intensity analysis of tumors and major organs 24 h after intravenous injection of GVs-IR808 in mice. (n = 3, p < 0.05); (I) Fluorescence microscope images of tumor sections at 24 h post-injection of GVs-IR808 + US, GVs-IR808 and PBS groups. Scale bar = 100 µm.

3.5. In vivo photothermal anti-tumor efficacy of GVs-IR808

Firstly, we detected the temperature elevation effect of GVs-IR808 in tumor by using of acoustic delivery of IR808 combined with laser irradiation. After 24 h of systemic administration of GVs-IR808 or PBS to tumor-bearing mice, acoustic irradiation was applied to the tumor under the guide of contrast imaging, followed by laser irradiation at 1.0 W/cm2 for 10 min. As can be seen from Fig. S5 and S6, the temperature of tumors in the GVs-IR808 + US group significantly increased, achieving 23.22 ± 1.73 °C ΔT within 10 min. In contrast, the tumor temperature of PBS-injected mice only increased about 5.44 ± 1.59 °C after 10 min of laser irradiation. Based on these data, we further evaluated the in vivo photothermal anti-tumor efficacy of GVs-IR808. Fig. 6A showed the in vivo experimental procedure. 4 T1 tumor-bearing mice were divided into 6 groups, including: the control group, US + Laser group, GVs-IR808 group, GVs-IR808 + US group, GVs-IR808 + Laser group and GVs-IR808 + US + Laser group. The tumor volume and body weight of mice were recorded at every other day. As expected, no significant tumor growth inhibition was observed in the control, US + Laser, GVs-IR808, and GVs-IR808 + US groups. In contrast, tumor growth was significantly inhibited in the GVs-IR808 + Laser group and GVs-IR808 + US + Laser group, suggesting that the acoustic delivery of IR808 via GVs-IR808 combined with laser irradiation showed significant anti-tumor efficacy. Notably, GVs-IR808 + US + Laser group produced the most significant anti-tumor effect, leading to complete tumor regression in all treated mice (Fig. 6B and 6C). No significant weight changes were observed in all groups of mice within 20 days (Fig. 6D). The survival time of mice in the GVs-IR808 + US + Laser group was also significantly prolonged in comparison with the other groups (Fig. 6E). H&E and TUNEL staining showed that significantly more tumor necrosis was observed in the GVs-IR808 + US + Laser group than in the other groups. Ki67 immunohistochemical staining also showed that treatment with GVs-IR808 + US + Laser significantly inhibited the proliferation of tumor cells. In contrast, no obvious tumor cell proliferation inhibition was observed in the US + Laser, GVs-IR808 and GVs-IR808 + US groups (Fig. 6F).

Fig. 6.

Fig. 6

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In vivo anti-tumor efficacy of GVs-IR808. (A) Schematic illustration of the in vivo experimental procedure of ultrasound cavitation-mediated PTT. (B) Photographs of 4 T1 tumor-bearing mice in the six groups during a 20-day period after various treatments; (C) Tumor growth curvesof six groups after treatment (n = 5, mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001); (D) Body weight changes of mice in six groups after various treatments (n = 5, mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001); (E) Percent survival of tumor-bearing mice after various treatments (n = 5); (F) Representative images of 4 T1 tumor sections by H&E staining, TUNEL staining and Ki-67 immunochemical staining. Scale bar = 100 µm. Here, U refers to ultrasound, and L refers to laser treatment.

3.6. Biosafety analysis of GVs-IR808

To evaluate the biosafety of GVs-IR808, the in vivo toxicity tests were performed on these treated mice. No abnormal behavior was observed in these mice over a period of 14 days. Moreover, they were sacrificed for histological and blood examination on the 14th day. No histological morphology changes were observed in the H&E-staining sections of the main organs including heart, liver, spleen, lung, and kidney (Fig. 7A). In addition, it was found negligible difference between GVs-IR808 and PBS-administrated mice in the typical liver and kidney function markers (such as ALT and BUN) and the red/white blood cells, confirming that the treatment of GVs-IR808 combined with acoustic delivery and laser irradiation had the favorable biosafety (Fig. 7B).

Fig. 7.

Fig. 7

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Biosafety analysis of GVs-IR808. (A) H&E sections (scale bar = 100 μm) of main organs (heart, liver, spleen, lungs and kidneys) of mice received with different treatments after 14 days; (B) Changes of blood biochemical indexes in each group with different treatments. Here, U refers to ultrasound, and L refers to laser treatment.

4. Discussion

Malignant tumors present a significant risk to the health and well-being of patients. Ultrasound targeted microbubble destruction (UTMD) has become a novel technology to locally deliver drugs or therapeutic agents into the diseased tissues. In this procedure, ultrasound contrast agents such as microbubbles and nanobubbles play a key role. They function as the cavitation nuclei and produce the sonoporation effects on the blood vessels or tumor cells, facilitating drugs or therapeutic agents to deliver into the diseased tissues or tumor cells. Generally, ultrasound contrast agents can be divided into two types on the basis of synthesis method: the one is the chemically synthesized bubbles and the other is the biological synthesized bubbles [16,17]. The chemically synthesized ultrasound contrast agents, such as SonoVue and Snonazoid, have been commercially available [18,19] and have a particle size of about 1–8 µm. Due to the microscale size, these bubbles are confined in the blood vessels [20,21], and thus are difficult to reach extravascular tissues or tumor cells[22], greatly reducing the cavitation efficiency of bubbles. In this study, we used gas vesicles (GVs) as the cavitation nuclei. GVs are a new kind of ultrasound contrast agents isolated from Halobacterium NRC-1, with a particle size of approximately 200 nm, Among all biosynthetic gas vesicles (GVs), those GVs from Halobacterium NRC-1 exhibit larger particle sizes and ball-like structure. These structural characteristics endow GVs with stronger ultrasound responsiveness and enhanced contrast-enhancing capabilities in ultrasonic imaging [23]. The nanoscale size of GVs facilitates them to pass through the tumor blood vessels via EPR effects and contact tumor cells. Upon receiving with acoustic irradiation, they can directly perforate the tumor cells, enhancing the cavitation effects and drug delivery efficiency.

In recent years, various types of NIR photothermal agents have emerged, such as metallic nanomaterials, organic dyes and semiconductor nanomaterials [[24], [25], [26]]. The natural indole anthocyanine dye IR808 possesses excellent photostability, strong water solubility and high fluorescence intensity, and is therefore considered to be an effective NIR photothermal agent suitable for fluorescence imaging and PTT of tumor, which can not only accurately determine the location of tumors, but also improve the accuracy of PTT treatment under the guide of fluorescence imaging. Therefore, in this study, we developed GVs-IR808 as a novel photothermal agent through conjugating IR808 onto the surface of GVs. GVs-IR808 have several obvious advantages including: 1) GVs-IR808 has excellent ultrasound imaging and fluorescence imaging performance, as shown in Fig. 5. 2) GVs-IR808 has nanoscale particle size, making it possible extravasate into tumor via EPR effects. This process can be visualized through ultrasound contrast imaging. 3) More importantly, IR808 can be acoustically delivered into tumor cells through GVs-based cavitation effects. Thanks to the fluorescence imaging capability of IR808, the IR808 delivered into tumor can be monitored and used to guide laser irradiation for PTT. In this study, the conjugation efficiency between GVs and IR808 was determined to be 43 %. This efficiency is consistent with results reported by serveral other research groups. The observed moderate efficiency may be attributed to suboptimal catalytic conditions and steric hindrance effects. To achieve higher conjugation yields, future studies should focus on systematic optimization of conjugation parameters. As shown in Fig. 3, our synthesized GVs-IR808 has good photothermal conversion ability and photothermal stability.

Currently, imaging-guided PTT has become a new strategy for tumor ablation or intraoperative adjuvant therapy due to its non-invasiveness, good specificity, effective tumor ablation and fewer side effects [27]. After laser irradiation, photothermal agents may convert light energy into heat energy, which cause a heat shock response (when the temperature reaches 41 °C) or rapid cell death due to protein denaturation and membrane disruption (upon reaching 60 °C)[28]. Therefore, the localization of laser beam in the tumor is very important for precise tumor ablation. After acoustic delivery of IR808 into the tumor, these delivered IR808 could be visualized by fluorescence imaging, guiding near-infrared to precisely localize at the tumor site, up to 55.1 °C of temperature increase was observed in tumor by infrared thermography (Fig. S4 and S5), which was sufficient to induce irreversible tumor damage as a PTT reagent. As shown in Fig. 6, the tumor growth in the GVs-IR808 + US + Laser group was significantly inhibited, producing the strongest anti-tumor efficacy.

5. Conclusions

In summary, we developed GVs-IR808 as a new photothermal agent for tumor therapy. GVs-IR808 possesses ultrasound/fluorescence dual-modal imaging capability, making it possible visualize GVs-IR808 to perfuse into the tumor by ultrasound. Upon they received with short pulse acoustic irradiation, acoustic cavitation effects would occur and effectively deliver IR808 into the tumor cells. The significantly enhanced IR808 delivery into tumor not only could be used for guiding laser beam to precisely irradiate the tumor, but also greatly improved the anti-tumor efficacy through PTT. Thus, our study provides a novel strategy for tumor PTT treatment.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Xin Meng: Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Formal analysis. Yanan Feng: Validation, Methodology. Yuanyuan Wang: Visualization, Data curation. Chen Lin: Methodology, Data curation. Wei Wang: Resources, Formal analysis. Sutian Zhu: Validation, Resources. Bing Guo: Validation, Supervision. Huaiyu Wang: Resources, Methodology. Litao Sun: Investigation, Formal analysis. Fei Yan: Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge the support of the National Key R&D Program of China (2020YFA0908800), Shenzhen Medical Research Fund (Grant No.D2301012, B2402006), the National Natural Science Foundation of China (32171365, 82071929, 82202158),the Key Research and Development Project of Vanguard and Leading Goose in Zhejiang Province (No.2024C03069), Guangdong Innovation Platform of Translational Research for Cerebrovascular Diseases.

Footnotes

☆

This article is part of a special issue entitled: ‘Biomaterial Assembly and Theranostics’ published in Ultrasonics Sonochemistry.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2025.107398.

Contributor Information

Litao Sun, Email: litaosun1971@sina.com.

Fei Yan, Email: fei.yan@siat.ac.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Data 1
mmc1.docx (2.4MB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data 1
mmc1.docx (2.4MB, docx)

Data Availability Statement

Data will be made available on request.

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